Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg numbers through TGF-?, MHC class II, and CCL22. In wild type mice, MFG-E8 restrains the potency of GM-CSF secreting B16 melanoma vaccines through Treg induction, while a dominant negative MFG-E8 mutant (RGE) potentiates therapeutic immunity through Treg inhibition. In patients, MFG-E8 is expressed at high levels in melanoma cells and/or tumor associated myeloid elements upon progression to the vertical growth phase. MFG-E8 acts as a melanoma promoter through coordinated ?v?3 integrin signaling in the tumor microenvironment, thereby stimulating melanoma cell resistance to apoptosis, epithelial-to-mesenchymal transition, invasion, angiogenesis, and immune suppression. Inhibition of MFG-E8 with shRNAs or systemic antibodies enhances the anti-tumor activity of cytotoxic treatments in vitro and in vivo. Together, our results suggest that MFG-E8 blockade may trigger melanoma destruction through both immune-mediated and tumor cell autonomous pathways. In this project, we will explore the mechanisms underlying the anti-tumor effects of antagonizing MFG-E8 using pre-clinical models and blood samples from patients. Our Specific Aims are: 1. To elucidate the biologic activities of GM-CSF/RGE secreting melanoma vaccines in murine models. 2. To elucidate the biologic activities of anti-MFG-E8 antibodies in murine melanoma models. 3. To characterize the human GM-CSF/MFG-E8/Treg pathway using melanoma patient samples. PUBLIC HEALTH RELEVANCE: This project explores a new therapeutic approach for malignant melanoma (the most lethal form of skin cancer) that involves targeting a protein called MFG-E8. We have shown that MFG-E8 functions to promote the growth of melanoma cells and to inhibit protective immune responses against melanoma. Blocking the actions of MFG-E8 in mouse models results in melanoma destruction. Work in this project will clarify the ways in which MFG-E8 blockade accomplishes melanoma killing, which in turn should support the development of MFG-E8 targeted treatments for melanoma patients.